Exercise intolerance is one of the clinical hallmarks of late-onset Pompe disease (LOPD). We studied the acute effects of ERT on the physiological variables associated with exercise tolerance in patients chronically ERT treated. Moreover, we assessed the influence of clinical severity on the investigated variables. The day before (B) and the day after (A) ERT injection, 11 LOPD patients performed on a cycle-ergometer an exercise tolerance test to voluntary exhaustion; VO2, HR, RPE, and GAA activity were determined in B and A. The disease severity was characterized by Walton scale, 6MWT, and pulmonary function tests. No significant differences in the variables related to exercise tolerance were found in A vs B, despite a significant increase in GAA activity in peripheral lymphocytes. No differences in VO2 peak were observed between patients with only skeletal muscle impairment and patients with both skeletal and respiratory muscle impairment. Distance walked at 6MWT was significantly higher than VO2 peak expressed as percentage of normal values. In conclusion, in LOPD patients the exercise tolerance test is not acutely affected by ERT administration; the peripheral muscle component seems more prominent in determining the VO2 peak decrease than the respiratory component; VO2 peak might be more sensitive than 6MWT in estimating exercise tolerance in LOPD.